Figure 1.

Human GPR109A is an N-glycosylated receptor. A) Four hours after the HeLa cells were transfected with empty vector (mock) or HA-tagged hGPR109A, they were pretreated with 3.0 μg/ml TM (+) or 1.0 μg/ml Bref-A (+), or were left untreated (−). After 20 h, the protein concentrations of cell homogenate supernatants were measured and separated using SDS-PAGE. After staining with anti-HA and horseradish peroxidase-conjugated anti-rat IgG as primary and secondary antibodies, respectively, the molecular masses of the expressed HA-tagged hGPR109A products were determined using Western blot analysis. B) Twenty-four hours after transfection with empty vector (mock) or HA-tagged hGPR109A, HeLa cells were homogenized and treated with Endo-H (+) or PNGase-F (+), or were left untreated (−). The molecular mass of hGPR109A was determined using SDS-PAGE and Western blot analyses, as described in the Materials and Methods. The positions of molecular markers are indicated on the right, and the approximate molecular sizes of hGPR109A are shown on the left. Data are representative of 3 independent experiments that yielded similar results.